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Context: Lipoid congenital adrenal hyperplasia (LCAH) is caused by mutations in STAR. Classic (CLCAH) and nonclassic (NCLCAH) forms were reported as total and partial deficiencies, respectively, of adrenal and gonadal steroid hormones. The rarity of LCAH has precluded large-scale epidemiological and clinical investigations., Objective: To determine the epidemiological and clinical characteristics of 2 forms of LCAH., Design: A multicenter cross-sectional cohort study in Japan on December 1, 2017., Participants: Fifty-seven patients with LCAH (median age, 23.7 years; range, 0.0-47.5 years)., Main Outcome Measures: Patient demographics, STAR genotype, Quigley grade, endocrinological and imaging data, treatment, and prognosis., Results: Fifty-three and 4 patients fulfilled definite and probable diagnostic criteria for LCAH, respectively. When NCLCAH was defined as either Quigley grade 1 in XY karyotype, no episode of salt losing or requirement of fludrocortisone, or onset of primary adrenal insufficiency (PAI) at 1 year or older, patients were divided into groups of 43 patients with CLCAH (75.4%), 11 with NCLCAH (19.3%), and 3 with unclassified LCAH (5.3%). All of the patients with CLCAH and 7/11 NCLCAH (63.6%) were treated with fludrocortisone. CLCAH was diagnosed at a significantly younger age than NCLCAH (median, 0.0 vs 4.0 years). STAR-Arg272Cys or -Met225Thr was identified only in NCLCAH (8/11, 72.7%)., Conclusions: We demonstrated the relative proportions and clinical and molecular characteristics of NCLCAH and CLCAH in Japan. These criteria for NCLCAH correspond to all previously published cases and our cases whose masculinization of the external genitalia, ability of mineralocorticoid production, and onset of PAI were described., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Maternal-fetal calcium (Ca 2+ ) transport in the placenta plays a critical role in maintaining fetal bone mineralization. Mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) have been identified as causative mutations of transient neonatal hyperparathyroidism due to insufficient maternal-fetal Ca 2+ transport in the placenta. In this study, we found two novel mutations in subjects that have transient neonatal hyperparathyroidism. TRPV6 carrying the mutation p.Arg390His that localizes to the outer edge of the first transmembrane domain (S1) showed impaired trafficking to the plasma membrane, whereas TRPV6 having the mutation p.Gly291Ser in the sixth ankyrin repeat (AR) domain had channel properties that were comparable those of WT channels, although the increases in steady-state intracellular Ca 2+ concentration could have led to Ca 2+ overload and subsequent death of cells expressing this mutant channel. These results indicate that the AR6 domain contributes to TRPV6-mediated maintenance of intracellular Ca 2+ concentrations, and that this region could play a novel role in regulating the activity of TRPV6 Ca 2+ -selective channels.

We report the case of a 43-year-old female patient who presented with symptoms of abnormal behavior, hearing loss, ataxic gait, central hyperventilation which had appeared over the course of one month. Brain MRI showed no abnormal findings in DWI and EEG did not indicate periodic synchronous discharge (PSD). Over the course of the same month, she also presented with central apnea that intermittently showed spontaneous improvement and reappearance. Cerebrospinal fluid 14-3-3 protein tested negative and there was no family history, but an abnormal prion protein was detected in the cerebrospinal fluid by the RT-QUIC assay. We diagnosed her with familial Creutzfeldt-Jakob disease (CJD) with an E200K mutation after genetic examination. Both high cortical signals on MRI and PSD on EEG were not recognized even in the advanced stage. Central apnea was presumed to be caused by disorders of the respiratory center of the brainstem. Hearing loss was also considered to be an obstacle at the brainstem level from the latency delay after the III wave in auditory brainstem response (ABR). The possibility of brain stem symptoms occurring in the early stages of CJD should be considered.

Purpose: Our sincere hope is to establish the predictive factors of neoadjuvant chemotherapy (NAC) response and provide patients with greater certainty regarding treatment outcomes. The aim of this study was to assess the response to NAC and survival in patients with locally advanced rectal cancer (LARC) according to their RAS/BRAF mutation status., Methods: Data on 57 patients with LARC who received NAC between 2009 and 2016 were analyzed retrospectively. The patients were classified into two groups based on their mutation status: wild-type in both RAS and BRAF (WT) or mutant-type in either RAS or BRAF (MT)., Results: Twenty-three patients were classified as WT, and the remaining 34 patients were MT. Histological response to NAC was similar in both groups. In responders, the 3-year relapse-free survival (RFS) was better compared with the non-responders (92 and 66%, respectively). In the WT group, the 3-year RFS was 95% which was significantly better than that in the MT group (59%, p = 0.011). The MT group was further subdivided into the following 2 groups by the pathological response; the MT responders (n = 10) and MT non-responders (n = 24). The 3-year RFS was 50% in the MT non-responders, which was significantly worse compared to that in the remaining patients (92%, p = 0.001)., Conclusion: RAS/BRAF mutations did not affect the response to NAC. However, the RFS was likely to be poor for those in the MT group who did not achieve favorable pathological response. In contrast, the RFS was favorable in the WT group regardless of the pathological response.

Background: Dual oxidase 2 (DUOX2) mutations are a cause of dyshormonogenesis (DH) and have been identified in patients with permanent congenital hypothyroidism (PH) and with transient hypothyroidism (TH). We aimed to elucidate the prevalence and phenotypical variations of DUOX2 mutations., Methods: Forty-eight Japanese DH patients were enroled and analysed for sequence variants of DUOX2, DUOXA2, and TPO using polymerase chain reaction-amplified direct sequencing., Results: Fourteen sequence variants of DUOX2, including 10 novel variants, were identified in 11 patients. DUOX2 variants were more prevalent (11/48, 22.9%) than TPO (3/48, 6.3%) (p=0.020). The prevalence of DUOX2 variants in TH was slightly, but not significantly, higher than in PH. Furthermore, one patient had digenic heterozygous sequence variants of both DUOX2 and TPO., Conclusions: Our results suggest that DUOX2 mutations might be the most common cause of both PH and TH, and that phenotypes of these mutations might be milder than those of other causes.

Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock-in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)-α production and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2(KI/KI) ). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2(KI/+) ) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders., (© 2014 American Society for Bone and Mineral Research.)

Purpose: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined., Methods: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced., Results: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR., Conclusion: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

Objective: SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model., Methods: CIA was induced in wild-type (Sh3bp2(+/+)) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2(KI/+)) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures., Results: Sh3bp2(KI/+) mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2(KI/+) mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2(+/+) and Sh3bp2(KI/+) cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2(+/+) and Sh3bp2(KI/+) mice. In vitro experiments showed that TNF-α production in Sh3bp2(KI/+) BMMs is elevated compared with Sh3bp2(+/+) BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2(KI/+) BMMs associated with increased NFATc1 nuclear localization., Conclusion: Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty. X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.

Marfan syndrome (MFS), an autosomal dominant connective tissue disorder, is caused by mutations in the gene encoding fibrillin 1 (FBN1). The clinical spectrum and severity of MFS disorder varies greatly both between and within families. Since there have been only a few reports on the relationship between FBN1 genotypes and clinical phenotypes in Japanese patients, the FBN1 gene was analyzed in 27 Japanese patients diagnosed with MFS. The nucleotide sequence of the 65 exons of the FBN1 gene was analyzed by PCR and direct sequencing. We have identified six polymorphisms and nine mutations including: four missense mutations (C1652Y, Q2054P, D2127Y, C2221R) in six patients, three nonsense mutations (R215X, S813X, R2220X) in three patients, and two frameshift mutations (2567insT, 7790insT) in three patients. Six of these nine mutations were in the calcium-binding epidermal growth factor-like domains all causative mutations detected except for C2221R were novel. It has been reported that the severe phenotypes of infantile MFS correlate with mutations in the mid region of FBN1, however, mutations were not detected in this region in the population analyzed in this study. Our results suggest that the location of the mutation is not the sole determinant of phenotypic severity; rather there is some difference in the genetic basis of MFS between Japanese and Caucasian populations., (Copyright 2001 Wiley-Liss, Inc.)

DNA polymerase beta (polbeta) is one of mammalian DNA polymerases and is known to be involved in a G:T/G:U mismatch repair. In order to investigate an involvement of this enzyme in a base excision repair, we searched a mutation of human polbeta in human gastric cancer and studied a function of the mutation. We observed cancer-specific missense mutations in 6 of 20 samples. All of these mutations were, however, heterozygous. We further analyzed the base excision repair activity of these mutants to know whether these mutants cause an error of mismatch repair. One of these mutants, which resulted in an amino acid substitution of Glu for Lys at codon 295, showed an inhibitory effect by in vitro base excision repair assay, suggesting that this mutation might play some role in carcinogenesis of the gastric mucosa.

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2, CDKN1C, and KVLQT1, are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A, BWR1B, and BWR1C. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O'Keefe, D., Dao, D. , Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021-2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith-Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/Cdk complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5 (ref. 2), a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a cancer-predisposing syndrome, making it a tumour-suppressor candidate. Several types of childhood tumours including Wilms' tumour, adrenocortical carcinoma and rhabdomyosarcoma exhibit a specific loss of maternal 11p15 alleles, suggesting that genomic imprinting is involved. Genetic analysis of the Beckwith-Wiedemann syndrome indicated maternal carriers, as well as suggesting a role of genomic imprinting. Previously, we and others demonstrated that p57KIP2 is imprinted and that only the maternal allele is expressed in both mice and humans. Here we describe p57KIP2 mutations in patients with Beckwith-Wiedemann syndrome. Among nine patients we examined, two were heterozygous for different mutations in this gene-a missense mutation in the Cdk inhibitory domain resulting in loss of most of the protein, and a frameshift resulting in disruption of the QT domain. The missense mutation was transmitted from the patient's carrier mother, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. Consequently, little or no active p57KIP2 should exist and this probably causes the overgrowth in this BWS patient.

The DAX-1 [DSS (dosage-sensitive sex)-AHC critical region in the X, gene 1] gene has been reported to be responsible for X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism. However, the function and structure of the DAX-1 protein have not been characterized. In this study, molecular analysis of the DAX-1 gene from 6 patients with AHC, including 2 siblings, identified 5 novel mutations with 3 nonsense mutations and 2 frameshift mutations. Case 1 had a nonsense mutation at position 395 (Q395X). Cases 2 and 3, who were siblings, had a nonsense mutation at position 91 (Y91X). Case 4 had a 2-base deletion (AT) at nucleotides 1610 and 1611 and a 1-base insertion (G) resulting in a premature stop codon at position 462 (1610-1611 del AT ins G). Case 5 had a nonsense mutation at position 271 (Y271X). Case 6 had a 1-base deletion (C) at nucleotide 1169, which induced a frame shift and a premature stop codon at position 371 (1169 del C). All mutated DAX-1 proteins had truncated C-terminal domains. In addition, reverse transcription-PCR and direct sequencing characterized the mutant messenger ribonucleic acid in testis from case 1. Our results suggest that these 5 novel mutations are responsible for X-linked AHC and that the C-terminus of the DAX-1 protein, especially the terminal 11 amino acids, is necessary for normal adrenal cortical embryogenesis.

Spontaneous mutations were accumulated for a total of 1,678,388 allele-generations in several hundred replicate second chromosome lines. These lines were compared of a Cy chromosome and one of three different lethal bearing chromosomes from natural populations. Electrophoretic mobility and/or activity change was screened for seven enzyme loci. Forty-four null mutations were detected, but no band-morph mutations were observed. There is significant variation in null mutation rate among enzyme loci and among lethal chromosomes. This may be attributable to differences in the distribution of transposable elements among the lines. The band-morph mutation rate is pooled with previous results and becomes 7.48 x 10(-7) with a 95% confidence limit of 2.04 x 10(-7) to 1.91 x 10(-6) per locus per generation. Similarly, the pooled null mutation rate becomes 1.30 x 10(-5) with 95% confidence limit of 1.15 x 10(-5) to 1.52 x 10(-5) per locus per generation.

Significant genetic variance in glycerol-3-phosphate dehydrogenase (GPDH) activity was observed between chromosome lines of Drosophila melanogaster that had each accumulated spontaneous mutations for approximately 300 generations. No restriction map variation was found in a 26-kb region surrounding the entire Gpdh gene. The restriction analysis used is capable of detecting insertions/deletions larger than 0.05 kb. The survey would also detect chromosomal recombinations that include the entire Gpdh coding region. Therefore, if the spontaneous mutations that affected the enzyme activity are located inside the Gpdh gene region, then they are base pair substitutions or structural changes that are smaller than the limit in resolution described above.

An experimental method is proposed for detecting the effects of positive natural selection on DNA polymorphisms. Since beneficial mutations are expected to increase in frequency faster than neutral mutations, variants which have reached high frequencies in a relatively short period could be linked to some beneficial mutation. D. melanogaster has a cosmopolitan polymorphic inversion -In(2L)t- whose age in some local populations has been estimated. Setting the age of In(2L)t as the upper limit for the age of variants, we searched for variants whose frequencies were possibly influenced by positive natural selection. We detected a single candidate whose frequency and distribution met the requirements imposed by our method.

The nucleotide sequence of a patient's aldolase B gene was determined and showed a substitution of a single nucleotide (C----A) at position 720 in the coding region, which resulted in the 240th amino acid, a cysteine, being changed to a stop codon (TGC----TGA). By an allele-specific oligonucleotide probe and polymerase chain reaction, the patient was shown to be homozygous for the mutation. To examine whether this mutation causes functional defect of the enzyme, the activity of the aldolase B from the patient, expressed in Escherichia coli by using expression plasmid, was measured. No activity was observed, and the predicted product was recovered from E. coli expression plasmid, indicating that this nonsense mutation was the cause of aldolase B deficiency.

The diversifying selection due to genotype-environment interaction can increase the genetic variation in natural populations. It is known, however, that the conditions for stable genetic polymorphism or marginal overdominance are quite restricted in this selection model. In this paper a simple model of diversifying selection was examined, and the following results were obtained: (1) Even when the conditions for marginal overdominance are not satisfied, if the diversifying selection is operating, the frequency of mutants can be higher than that in the case of simple mutation-selection balance. (2) This selection model causes a large amount of genetic load (environment load), even when the conditions for marginal overdominance are not satisfied, namely even when the equilibrium frequency of mutant is very low. From these results it can be concluded that the number of loci on which this type of diversifying selection is operating is very small, if any.

A considerable proportion of visible mutations is reported to be caused by the insertion of mobile genetic elements in Drosophila and other organisms. We estimated transposition rates of some Drosophila mobile elements by using the lines AW and JH in which spontaneous mutations have been accumulated independently for about 400 generations. Occupied sites of the mobile elements were detected by in situ hybridization on the salivary gland chromosomes sampled from 40 AW and 30 JH lines. The rates of insertion and excision of the copia and two copia-like elements, 412 and 17.6, are very low: Insertions occurred at up to 10(-3) per second chromosome per generation (17.6) and excision occurred at about 10(-5) per site per generation (copia and 412). Insertions of the I and hobo elements occurred much more frequently. These estimates are not only important for assessing the actual rate of various types of mutations but also for developing an evolutionary theory of mobile elements themselves.

The lethal and detrimental loads per second chromosome rapidly increased from 1968 to 1970 in a local population of Drosophila melanogaster in Japan (lethal load, from about 0.16 to 0.38; detrimental load, from 0.125 to 0.231 [Watanabe, T. K., Watanabe, T. & Oshima, C. (1976) Evolution 30, 109-118]). When the homozygous loads were measured in 1983, the lethal load had decreased to approximately the original amount (0.19) but the detrimental load had stayed high (0.241). The rise and fall of the lethal load can be accounted for by a P-type element that invaded a population with M cytotype, producing a high mutation rate. The mutation rate fell back to the earlier value after the cytotype became P. That the detrimental load did not decrease can be explained by assuming a longer persistence for detrimental mutations in the population. Evidence for a P-type mutator factor is that the mutation rate of the wild-type chromosomes differs between the different cytoplasmic and chromosomal backgrounds, being lower in the background from which the chromosomes were taken.

In a marked-inversion-balanced lethal system mutations were accumulated at a minimum pressure of natural selection on 2000 second chromosomes of Drosophila melanogaster that originated from 4 stem chromosomes. Five enzyme loci were tested: alpha-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), malate dehydrogenase (Mdh, EC 1.1.1.37), alcohol dehydrogenase (EC 1.1.1.1), hexokinase-C (Hex-C tentative name), and alpha-amylase (Amy, EC 3.2.1.1). Three band-morph mutants, one at the Mdh locus, one at the Hex-C locus, and one at the Amy locus, were detected out of 1,658,308 allele replications. In addition, 17 null mutants were found. Accepting that the number of structural genes is the same as that of bands in the salivary gland chromosomes, the total mutation rate per generation for all the structural genes in the second chromosomes is estimated to be 0.008-0.040, which is much smaller than that estimated for viability polygenes (0.12-0.17). Thus, it is speculated that most viability and other fitness polygenes are located in controlling regions outside the structural genes.